Electroporation is a recently identified procedure by which nonpermeant molecules may be introduced into the cell through the cell wall using an electric field. A prior art patent purporting to achieve this effect with respect to DNA is that of T. Wong, U.S. Pat. No. 4,849,355. In this, as in other electroporation procedures discussed in the literature, the cells to be electroporated were held in suspension in an electrolytic solution.
Experience to date has shown that while the rate of cell penetration can be improved by selecting the applied electrical voltage, as the voltage increases, a point is reached where cell mortality increases substantially. Apparently, successful electroporation, in terms of elevated the percentage of cells into which the desired molecules are introduced, combined with minimal cell damage, occurs only over a narrow range of applied voltages. More precisely, it is assumed that it is the electrical field strength, in volts per centimeter in the vicinity of the subject cells that is a major parameter in achieving successful electroporation, or the electrical current that corresponds to such electrical field.
In one article, "Electroporation and DNA Transfer" by Joyce L. Knutson and Daniel Yee, Analytical Biochemistry 164, 44-52 (1987), reference is made to the intensity of the electrical field as a parameter governing successful electroporation. Pulsed electric fields having momentary strengths on the order of 3.5 kV/cm and 8 kV/cm are recited as having been used by earlier researchers. In this specific article field strengths of around 390-1060 V/cm were applied.
The electroporation chamber described by Knutson and Yee consisted of 1 cm wide aluminum foil electrodes, 1 cm apart. Presumably the field in this set-up was calculated by dividing the applied voltage, during discharge, by the separation distance of the electrodes, and by assuming that the phosphate-buffered saline solution forming the electrolyte in the electroporation chamber was amorphous.
Further this article hypothesizes that with a constant field existing across an electroporation chamber, the voltage drop across each cell in suspension is concentrated across the relatively non-conductive cell membrane. Thus this article supposes that break-down of the cell membrane of the mammalian-type cells being tested occurs when the voltage drop across the cell membrane is around 200 mV. It is thereby supposed that "pores" effective for electroporation occur under similar voltage conditions.
Accordingly, as is apparent from this and other articles in the literature, control over the strength of the electric field to which cells are exposed, or the electrical current equivalent, is considered to be an important parameter for successful electroporation.
All of the work on electroporation so far cited in the literature except Chang deals with cells in suspension, that is either cells normally growing as a suspension, such as cells of hemopoietic origin, or cells which normally grow while attached to a solid support (cells of fibroblast or epithelial origin) but which have been detached for the occasion through the use of proteolytic enzymes or EDTA (Ethylenediamine tetraacetate) to place them in suspension This detachment, however, disturbs cellular metabolism in general and especially the cell division cycle. This latter effect is shown by a substantial delay in cell entry into the S (DNA synthesis) phase in the case of cells which have been detached. Therefore, a preferred procedure for electroporation of adherent cells is to apply the electric field while the cells are attached to their solid growth surface.
This arrangement has the advantage over conventional procedures in that naturally adherent cells need not be treated with a proteolytic enzyme, such as trypsin, in order to lift them into suspension for electroporation treatment. This avoids the disruption of cell physiology, especially with regard to their division cycle, inherent in their detachment, and allows studies of the effects of introduced-molecules to proceed without the complications associated with such disruption.
One method of electroporating cells while they adhere to a surface is to grow such cells on one of the electrodes of the electroporation chamber. For convenience of observing such cells under a microscope a transparent substrate with a transparent electrically conductive surface may be employed as such an electrode. A disadvantage of using an electrode of this form is that, due to electrical resistance, a voltage drop may develop over the area of the conductive surface, particularly where a thin conductive film is laid over a non-conducting glass or plastic substrate. This voltage drop will cause progressive regions of the cell population on the surface of the electrode to be exposed to electrical fields of differing intensity. This may result in only a portion of the cell population being exposed to fields of preferred intensity.
The foregoing problem is premised on the use of a conductive surface that exhibits significant resistive loss when employed as an electrode in an electroporation chamber. A converse problem arises when an electrode of low resistivity is employed for such surface, and it is desired to expose a surface population of adherent cells to an electric field which varies in a controlled manner over the area of such surface.
While recognition has been given to the significance of the strength of the applied field as a factor affecting successful electroporation, no attempts have been made to create a generally uniform electrical field over a surface carrying a population of living adherent cells that are proposed to be electroporated. Further no one has arranged to expose a surface population of cells to an electroporating field which varies, along the surface in a continuous, geometrically regular fashion.
It is, therefore, an object of this invention to create uniform field strength conditions over a surface carrying adherent cells so that a larger number of cells may be treated simultaneously to essentially the same field strength, and thereby more closely control the successful electroporation of living cells.
A further object of this invention is to provide an electrical field of continuous and of systematically varying strength, in a geometric sense, for electroporating a population of adherent cells on a surface, and thereby permit a direct comparison of the response of such cells to being electroporated under local electric fields of differing strengths.
These, and further features of the invention will be more apparent from the descriptions which no follow.